4,7-dichlorofluorescein dyes as molecular probes

ABSTRACT

Long wavelength, narrow emission bandwidth fluorescein dyes are provided for detecting specially overlapping target substances. The dyes comprise 4,7-dichlorofluorescein, and particularly 2′, 4′,5′,7′-tetrachloro-4,7-dichloro-5-(and 5-) carboxyfluoresceins. Methods and kits for using the dyes in DNA analysis are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/949,444filed Sep. 7, 2001, which is a continuation of application Ser. No.09/580,754 filed May 30, 2000, now Pat. No. 6,403,812 which is acontinuation of application Ser. No. 09/273,655 filed Mar. 23, 1999, nowPat. No. 6,096,723 which is a continuation of application Ser. No.08/905,855 filed Aug. 4, 1997, now Pat. No. 5,885,778, which is acontinuation of application Ser. No. 08/400,780 filed Mar. 8, 1995, nowPat. No. 5,654,442 which is a continuation of application Ser. No.07/939,813 filed Sep. 3, 1992, abandoned, which is acontinuation-in-part of application Ser. No. 07/436,455 filed Nov. 14,1989, now Pat. No. 5,188,934, which are all incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates generally to fluorescent labelling techniques, andmore particularly, to the use of 4,7-dichlorofluosceins for detectingmultiple target substances in the same sample.

BACKGROUND

Many diagnostic and analytical techniques require that multiple targetsubstances in the same sample be labelled with distinguishablefluorescent tages, e.g. Lanler at al. J. Immunol., Vol. 132, pgs.151-158 (196)(flow cyometry): Gray et al. Chromosoma, Vol. 73. pgs. 9-27(1979) (flow system kayrotyping); Fung at al. U.S. Pat. No. 4,055,255(DNA sequencing); and Mayrand et al, Applied and TheoreticalElectrophoresis, Vol. 3, pgs. 1-11 (1992) (analysis ofelectrophoretically separated polymerase chain reaction (PCR) product).This requirement is particularly difficult to satisfy in DNA sequenceanalysis where at least four spectrally resolvable dyes are needed inmost automated sequencing approaches.

Presently, there are two basic approaches to DNA sequence determination:the dideoxy chain termination method. e.g. Sanger et al., Proc. Natl.Acad. Sci. Vol. 74, pgs. 5463-5467 (1977): and the chemical degradationmethod, e.g. Maxam et al, Proc. Natl. Acad. Sci. Vol. 74, pgs.569-564(1977). The chain termination method has been improved in several ways,and serves as the basis for all currently available automated DNAsequencing machines, e.g. Singer et al. J. Mol. Biol. Vol. 143. pgs.161-178 (1980); Schmier et al. J. Mol. Biol. Vol. 129, pp. 169-172(1979); Smith et al. Nucleic Acids Research Vol. 13, pgs. 2399-2412(1985): 35Smith et al, Nature, Vol. 321. pgs. 674-479 (1987); Prober etal, Science, Vol. 238, pgs. 338-341 (1987) Section II, Meth. Enzyml.,Vol. 155. pgs. 51-334 (1987): Church et al, Science, Vol 240, pgs.185-188 (1988): and Connell et al. Biotechniques, Vol. 5, pgs. 342-348(1987).

Both the chain termination and chemical degradation methods require thegeneration of one or more sets of labeled DNA fragments, each having acommon origin and each terminating with a known base. The set or sots offragments must then be separated by size to obtain sequence information.In both methods, the DNA fragments are separated by high resolution gelelectrophoresis. In most automated DNA sequencing machines, fragmentshaving different terminating bases are labeled with differentfluorescent dyes, which are attached either to a primer, e.g. Smith atal (1987, cited above), or to the base of a terminal dideoxynucleotide,e.g. Prober at al (cited above). The labeled fragments are combined andloaded onto the same gel column for electrophoretic separation. Basesequence is determined by analyzing the fluorescent signals emitted bythe fragments as they pass a stationary detector during the separationprocess.

Obtaining a set of dyes to label the different fragments is a majordifficulty in such DNA sequencing systems. First, it is difficult tofind three or more dyes that do not have significantly overlappingemission bands, since the typical emission band width for organicfluorescent dyes is about 4080 nanometers (nm) and the width of thevisible strum is only about 350400 nm. Second, even when dyes withnon-overlapping emission bands are found, the set may still beunsuitable for DNA sequencing if the respective fluorescent efficienciesare too low. For example, Pringle et al, DNA Core Facilities Newsletter,Vol. 1, pgs. 15-21 (1988), present data indicating that increased gelloading cannot compensate low fluorescent efficiencies. Third, whenseveral fluorescent dyes are used concurrently, excitation becomesdifficult because the absorption bands of the dyes are often widelyseparated. The most efficient excitation occurs when each dye isIlluminated at the wavelength corresponding to its absorption bandmaximum. When several dyes are used one is often forced to make a tradeoff between the sensitivity of the detection system and the increasedcost of providing separate excitation sources for each dye. Fourth, whenthe number of differently sized fragments in a single column of a gel isgreater than a few hundred, the physiochemical properties of the dyesand the means by which they are linked to the fragments becomecritically Important. The charge, molecular weight, and conformation ofthe dyes and linkers must not adversely affect the electrophoreticmobilities of closely sized fragments so that extensive band broadeningoccurs or so that band positions on the gel become reversed, therebydestroying the correspondence between the order of bands and the orderof the bases in the nucleic acid whose sequence is to be determined.Finally, the fluorescent dyes must be compatible with the chemistry usedto create or manipulate the fragments. For example, in the chaintermination method, the dyes used to label primers and/or the dideoxychain terminators must not interfere with the activity of the polymeraseor reverse transcriptase employed.

Because of these severe constraints only a few sets of fluorescent dyeshave been found that can be used in automated DNA sequencing and inother diagnostic and analytical techniques, e.g. Smith et al (1985,cited above); Prober et al (cited above); Hood et al, European patentapplication 8500960: and Connell et al (cited above).

in view of the above, many analytical and diagnostic techniques such sDNA sequencing, would be significantly advanced by the availability ofnew fluorescent dyes (1) which are physiochemically similar to readilyavailable dyes, (2) which permit detection of specially overlappingtarget substances, such as closely spaced bands of DNA on a gel, (3)which extend the number of bases that can be determined on a single gelcolumn by current methods of automated DNA sequencing, and (4) which areamenable for use with a wide range of preparative and manipulativetechniques.

SUMMARY OF THE INVENTION

The invention is directed to a method of concurrently detectingspecially overlapping target substances using 4,7-dichlorofluoresceindyes, and in particular, methods of DNA sequence determination employing4,7-dichlorofluorescein dyes. The invention also includes2′,7′-dichloro-5 (and 6-) carboxy-4,7-dichlorofluorescein defined byFormula I.

wherein:

-   -   A′ is hydrogen, fluoro, chloro, a linking functionality, such as        isothiocyanate, succinimidyl carboxylate, or phosphoramidite, or        a group, such as carboxyl, sulfonyl, or amino, that may be        converted to a linking functionality: preferably A′ is a linking        functionality or a group that may be converted to a linking        functionality;    -   X′ is hydrogen, fluoro or chloro, such that whenever A′ is a        substituent of the 6 carbon atom X′ is a substituent of the 5        carbon atom, and whenever A′ is a substituent of the 5 carbon        atom X′ is a substituent of the 6 carbon atom; preferably, X′ is        hydrogen;    -   Z₃ is hydrogen, fluoro, chloro, a linking functionality, such as        isothiocyanate, succinimidyl carboxylate, or phosphoramidite, or        a group, such as carboxyl, sulfonyl, or methylamino, that that        may be converted to a linking functionality; preferably, Z₃ is        hydrogen or chloro;    -   Z₄ is hydrogen, fluoro, chloro, a linking functionality, such as        isothiocyanate, succinimidyl carboxylate, or phosphoramidite, or        a group, such as carboxyl, sulfonyl, or methylamino, that may be        converted to a linking functionality; preferably, Z₄ is hydrogen        or chloro;    -   B′ is fluoro, chloro, or an acidic anionic group; preferably, B′        is carboxyl or sulfonyl, and most preferably B′ is carboxyl;    -   and wherein at least one of A′, Z₃, and Z₄ is a linking        functionality or a group that may be converted to a linking        functionality. Preferably, only one of A′, Z₃ and Z₄ is a        linking functionality or a group that may be converted to a        linking functionality.

The invention also includes kits for carrying out the method of theinvention. Generally, kits are provided for detecting a plurality ofelectrophoretically separated classes of DNA fragments. In particular,kits are included for carrying out DNA sequencing wherein at beat oneclass of primer extension product is fluorescently labelled with a4,7-dichlorofluorescein dye. Such DNA sequencing kits include kits withdye-labelled primers and, as an alternative embodiment, kits withdye-labelled terminators.

Throughout, the Colour index (Association of Textile Chemists, 2nd Ed.,1971) carbon numbering scheme is used, i.e. primed numbers refer tocarbons in the xanthene structure and unprimed numbers refer to carbonsin the 9′-phenyl.

The invention is based in part on the discovery that the fluorescentproperties of 4,7-chloro-5-(and 6-)carboxyfluorescein and related dyesare highly favorable for use as molecular probes. Their emission bandwidths are generally 20-30 percent narrower than analogs lacking the4,7-dichloro derivatives, their emission and absorption maxima are atwavelengths generally about 10-30 nm high r than analogs lacking the4,7-dichloro derivatives, and their fluorescent efficiencies are high,in some cases being nearly triple those of analogs lacking the4,7-dichloro derivatives.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the invention is based in part on the discovery of aclass of fluorescein dyes that have absorption and emission maxima atunusually long wavelengths, narrow emission band widths and otherfavorable fluorescent properties. In addition, the invention includesthe novel fluorescein analogs defined by Formula I as members of thisclass of dyes. These dyes permit the assembly of novel sets ofspectrally resolvable, physiochemically similar dyes particularly usefulin automated DNA sequence analysis.

As used herein the term imperially resolvable in reference to a set ofdyes means that the fluorescent emission bonds of the dyes aresufficiently distinct, i.e. sufficiently non-overlapping, that targetsubstances to which the selective dyes are attached. e.g.polynucleotides, can be distinguished on the basis of the fluorescentsignal generated by the respective dyes by standard photodetectionsystems, e.g. employing a system of band pass filters andphotomultiplier tubes, or the like, as exemplified by the systemsdescribed in U.S. Pat. Nos. 4,230,558, 4,811,218, or the like, or inWheeless et al. pgs. 21-76, in Flow Cyometry:. instrumentation and DataAnalysis (Academic Pros, Now York, 1985)

The term “lower alkyl” as used herein directly or in connection withethers denotes straight-chain and/or branched chain alkyl groupscontaining from 1-6 carbon atoms. e.g. the term includes methyl, ethyl,propyl, isopropyl, tert-butyl, and the like. More preferably, the term“lower alkyl” denotes an alkyl having from 1 to 3 carbon atoms.

The term “halo” as used herein denotes the halogen atoms fluorine,chlorine, bromine, and Iodine; more preferably, the term denotesfluorine or chlorine; and most preferably, the term denotes chlorine.

Preferably, the 4,7-dichloro-5- (and 6-) carboxyfluorescein dyes of theinvention include those defined by Formula II.

wherein:

-   -   A′, B′ and X′ are defined as above;    -   Z₁ hydrogen or, when taken with Z₂, benzo;    -   Z₂, when taken alone, is hydrogen, halo, lower alkyl, lower        alkyloxy, or a group, such as carboxyl, sulfonyl, or        methylamino, that may be converted to an active linking        functionally, or when taken with Z₁, Z₂ is methylamino, that may        be convened to an active linking functionally, or when taken        with Z₅, Z₆ is benzo; preferably, when taken alone. Z₆ is        hydrogen, methyl, ethyl, fluoro, chloro, methoxy, or ethoxy.    -   and wherein at least one of A, Z₂, Z₃, Z₄, and Z₅ is a group        that may be converted to an linking functionality. Preferably,        only one of A, Z₂, Z₃, and Z₄, and Z₅ is a group that may be        converted to an active linking functionality.

Many dyes for use in the invention are commercially available or can besynthesized by techniques known in the art, e.g. Ghatak et al. J. Ind.Chem. Soc., Vol. 6. pgs. 471 (1929); and Khanna et al. U.S. Pat. No.4,439,356. Alternatively, fluorescein analogs, i.e. A=B=carboxyl, can besynthesized by reacting substituted resorcinol with substitutedbenzophenone or with substituted trimellitic acid in the presence ofpropionic acid, as illustrated in the examples. Sulfonylfluorosceins,i.e. A or B is sulfonyl, are synthesized following the methods disclosedby Lee et al. Cytometry. Vol. 10, pgs. 151-16 (1989), modified bysubstituting appropriate reactants to give 5- or 6-carboxyl orsulfonylfluoescein products. Preferably, when labelling polynucleotidesin DNA sequencing the 5- and 6-isomers of the dyes are used separatelybecause they typically have slightly different electrophoreticmobilities that can lead to band broadening if mixtures of the isomersused. The 5 and 6 lissome of the dyes are readily separated by reversephase HPLC, e.g. Edmundson et al. Mol. Immunol., Vol. 21, pg. 561(1194). Generally, it is believed that the flit eluting peak is the6-isomer and the second eluting peak is the 5-isomer.

Dyes of the invention can be attached to target substances by a varietyof means well known in the art. For example, Haugland. Handbook ofFluorescent Probes and Research Chemicals (Molecular Probes, inc.Eugene. 1989) provides guidance and examples of means for linking dyesto target substances. Substituent A is converted to a linkingfunctionally that can be reacted with a complementary functionality on atarget substance to form a linking group. The following table listslinking functionalities that can be formed whenever A is carboxyl,sulfonyl or amino, suitable complementary function and the resultinglinking groups suitable for use with the invention. Com- Linkingplementary Linking Functionality Functionality Group

Preferably the linking functionality k isothiocyanate, sulfonylchloride, 4,6-dichlorotriazinylamine, or succinimidyl carboxylatewhenever the complementary functionality is amine. And preferably thelining functionality is maleimide, or iodoacetamide whenever thecomplementary functionality is sulfhydryl. Succinimidyl carboxylates canbe formed by condensing the 5- and/or 6-carboxyls of the above dyes-with N-hydroxysuccinimide using dicyclohexylcarbodiimide (DCC). e.g. asillustrated in examples 6 and 8 of Khanna et al, U.S. Pat. No.4,318,848, and Kasai et al, Anal. Chem., Vol. 47, pgs. 3437 (1975).Accordingly, these references are incorporated by reference. Dyephosphoramidites are formed as taught by Stein et al. Gene, Vol. 72,pgs. 333-341 (1988); Fung at of, U.S. Pat. No. 4,757,141; Europeanpatent application 89118946.8 filed 13 Sep. 1989; and European patentapplication 88307934.5 filed 26 Aug. 1988. Substitutes R₁, R₂, and R₃can take a variety of forms, e.g. as taught by Beaucage et al.Tetrahedron, Vol. 48, pgs. 2223-2311 (192; Canthers, pgs. 47-94 inNorang, editor, Synthesis and Applications of DNA and RNA (cademicPress, New York, 1987); and the like. Preferably, R₁ and R₂, takenseparately, are methyl, ethyl, or isopropyl, and R₁ and R₂, takentogether with the nitrogen to which they are attached, is a heterocyclehaving from four to eight carbon atoms and one to two heteroatomsselected from the group consisting of nitrogen, oxygen, and sulfur. Morepreferably. R₁ and R₂, taken together with the nitrogen to which they amattached is morpholino. Preferably, R₃ is selected from the groupconsisting of methyl, chlorophenyl, β-cyanoethyl, methylsulfonylethyl,and nitrophenylethyl. Preferably, the phosphoramidite-derived linkinggroup is oxidized to form a phosphorus(V) linkage, e.g. as taught byBeaucage et al (cited above); Stec et al, PCT applicationPCTIUS91/01010; Beaucage et al, U.S. Pat. No. 5,003,097; or the like.

When dyes of the invention are used to label dideoxynucleotides for DNAsequencing, preferably they are linked to the 5 carbon of pyrimidinbases and to the 7 carbon of 7-deazapurine bases. For example, severalsuitable base labeling procedures have been reported that can be usedwith the invention, e.g. Gibson et al, Nucleic Acids Research, Vol. 15.pgs. 8455647 (1987); Gebeyehu et al. Nucleic Acids Research. Vol. 15.pgs. 4513-4535 (1987); Haralambidis et al, Nucleic Acids Research, Vol.15, pgs. 48584876 (1987); and the like. Preferably, the linking groupbetween the dye and a base is formed by reacting an N-hydroxysuccinimide(NHS) ester of a dye of the invention with an alkynylamino-derivatizedbase of a dideoxynudeotide. Preferably, the linking group is3carboxyamino-1-propynyl. The synthesis of such alkynylamino-derivatizeddideoxynudeotide is taught by Hobbs et al in European patent applicationnumber 87305844.0 and U.S. Pat. No. 5,047,519, which are incorporatedherein by reference. Briefly, the alkynylamino-derivatizeddideoxynucleotides are formed by placing the appropriatehalodideoxynucleoside (usually 5-iodopyrimidine and 7-Iodo-7-deazapurinedideoxynudeosides as taught by Hobbs et al (cited above)) and Cu(I) in aflask, flushing with Ar to remove air, adding dry DMF, followed byaddition of an alkynylamine, triethylamine and Pd(0). The reactionmixture can be stirred for several hours, or until thin layerchromatography indicates consumption of the halodideoxynucleoside. Whenan unprotected alkynylamine is used, the alkynylamino-nucleoside can beisolated by concentrating the reaction mixture and chromatographing onsilica gel using an eluting solvent which contains ammonium hydroxide toneutralize the hydroxyhalide generated in the coupling reaction. When aprotected alkynylamine is used, methano/methylene chloride can be addedto the reaction mixture, followed by the bicarbonate form of a stronglybasic anion exchange resin. The slurry can then be stirred for about 45minutes, filtered, and the resin rinsed with additional methoxymethyl,chloride. The combined filtrates can be concentrated and purified byflash-chromatography on silica gel using a methanol-methylene chloridegradient. The triphosphates are obtained by standard techniques.

Target substances of the invention can be virtually anything that thedyes of the invention can be attached to. Preferably the dyes arecovalently attached to the target substances. Target substances includeproteins, polypeptides, peptides, polysaccharides, polynucleotides,lipids, and combinations and assemblages thereof, such as chromosomes,nuclei, living cells, such as bacteria, other microorganisms, andmammalian cells, tissues, and the like. As used herein the term“polynucleotide” means a single stranded or double stranded chain of DNAor RNA in the size range of a few bases in length to several thousandbases in length. e.g. from 6 to a few tens to several hundreds or toseveral thousands of bases in length (of single stranded), or in thesize range of a few basepairs in length to several thousand basepairs inlength, e.g. from 8 to a few tens to several hundred or to severalthousand basepairs in length (of double stranded).

A number of complementary functionalities can be attached to the 5′ or3′ ends of synthetic oligonucleotides and polynucleotides, e.g. aminogroups, Fung et al, U.S. Pat. No. 4,757,141 and Miyoshi et al, U.S. Pat.No. 4,805,735; or sulfhydryl groups, Connolly, Nucleic Acids Research,Vol. 13, pgs. 4485-4502 (1985), and Spoat et al, Nucleic Acids Research.Vol. 15, pgs. 4837-4848 (1987).

Dyes of the invention are particularly well suited for identifyingClasses of polynucleotides that have been subjected to a biochemicalseparation procedure, such as gel electrophoresis, where a series ofbands or spots of target substances having similar physiochemicalproperties, e.g. size, conformation, charge, hydrophobicity, or thelike, are present in a linear or planar arrangement. As used herein, theterm “bands” includes any special grouping or aggregation of targetsubstance on the basis of similar or identical physiochemicalproperties. Usually bands arise in the separation of dye-polynucleotideconjugates by electrophoresis, particularly gel electrophoresis.

Classes of polynucleotides can arise in a variety of contexts. Forexample, they can arise as products of restriction enzyme digests, or asextension products in polymerase or “gas reactions. Preferably, classesidentified in accordance with the invention are defined in term: ofterminal nucleotides so that a correspondence is established between thefour possible terminal bases and the members of a set of spectrallyresolvable dyes. Such sets are readily assembled from the dyes of theinvention by measuring emission and absorption bandwidths withcommercially available spectrophotometers. More preferably, the classesarise in the context of the chemical or chain termination methods of DNAsequencing, and most preferably the classes arise in the context of thechain termination method. In either method dye-polynucleotide conjugatesare separated by standard gel electrophoretic procedures, e.g. Gould andMatthews, cited above; Rickwood and Hames, Eds., Gel Electrophoresis ofNucleic Acids: A Practical Approach, (IRL Press United, London, 1981);or Osterman, Methods of Protein and Nucleic Acid Research, Vol. 1(Springer-Verlag, Beriln, 1984). Preferably the type of gel ispolyacrylamide having a concentration (weight to volume) of betweenabout 2-20 percent. More preferably, the polyacrylamide gelconcentration is between about 4-8 percent. Preferably the gel includesa strand separating, or denaturing, agent. Detailed procedures forconstructing such gels are given by Maniatis et al., “Fractionation ofLow Molecular Weight DNA and RNA in Polyacrylamide Gels Containing 98%Formamide or 7 M Urea,” in Methods in Enzymoloay, Vol. 65, pgs. 299-305(1980); Maniatis et 91., “Chain Length Determination of Small Double-and Single-Stranded DNA Molecules by Polyacrylamide GelElectrophoresis,” Biochemistry Vol. 14. pgs. 3787-3794, (1975); andManiatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, New York, 1982). pgs. 179-185. Accordingly thesereferences are incorporated by reference. The optimal gel concentration,pH. temperature, concentration of denaturing agent, etc. employed in aparticular separation depends on many factors, including the size rangeof the nucleic acids to be separated, their base compositions, whetherthey are single stranded or double stranded, and the nature of theclasses for which information is sought by electrophoresis. Accordinglyapplication of the invention may require standard preliminary testing tooptimize conditions for particular separations. By way of example,polynucleotides having sizes in the range of been about 20-300 baseshave been separated and detected in accordance with the invention in thefollowing gel: 6 percent polyacrylamide made from 19 parts to 1 partascytamid to bacyamide, formed in a Triborate EDTA buffer at PH 8.3(measured at 25° C.) with 48 percent (weight/volume) urea. The gel wasrun at 50° C.

The dye-polynucleotide conjugates on the gel are illuminated by standardmeans, e.g. high intensity mercury vapor lamps, lasers, or the like.Preferably, the dye-polynucleotides on the gel are illuminated by laserlight generated by a argon ion laser, particularly the 488 and 514 nmemission lines of an argon Ion laser. Several argon ion lasers areavailable commercially which is simultaneously at these lines, e.g.Cyonics, Ltd. (Sunnyvale, Calif.) Model 2001. or the like.

in the chain termination method, dyes of the invention can be attachedto either primers or dideoxynucleotides. Dyes can be linked to acomplementary functionality on the 5′ end of the primer, e.g followingthe teaching in Fung et al, U.S. Pat. No. 4,757,141 which isincorporated herein by reference; on the base of a primer, e.g.following the teachings of Ward et al, U.S. Pat. No. 4,711,955; directlyto the 5′-hydroxyl via a phosphoramidite linking functionality: or onthe base of a dideoxynudeotide, e.g. via the alkynylamino linking groupsdisclosed by Hobbs et al, European patent application number 87305844.0which is incorporated herein by reference.

Kits of the invention can take a variety of forms, but usually pride themeans for the fluorescent detection of multiple DNAs separated by size.Kits may be used for detecting amplified nucleic acids separated by size(e.g. by electrophoresis), for DNA sequencing, and the like. Generally,the kits will include either an oligonucleotide labelled with a4,7-dichlorofluorescein dye, or in an embodiment of the DNA sequencingkit a dye-terminator mix wherein at least one of the dye-terminators islabelled with a 4,7-dichlorofluorescein dye. Usually, the dye-terminatoris a dideoxynucloside triphosphate, as d scribed above, labelled with afluorescent dye.

Kits for detecting amplified nucleic acids comprise at least oneoligonucleotide labelled with a 4,7-dichlorofluorescein dye, an enzymeselected from the group consisting of nucleotide polymerase and nucleicacid ligase, and a reaction buffer. Whenever the kit includes a DNApolymerase, it further includes a nucleoside triphosphate mix, e.g. a 50mM aqueous solution of EDTA containing the appropriate concentration ofnudeoside triphosphates for a particular application, e.g.amplification, sequencing, or the like. When the kit provides anucleoside triphosphate mix for DNA sequencing it is understood thatsuch triphosphates include analogs, such asnucleoside-5′-O-(1-thiotriphosphates), e.g. as taught by Lee et al,Nucleic Acids Research, Vol. 20, pgs. 2471-2483 (1992). Nucleic acidpolymerases include DNA polymerases, RNA polymerases, and reversetranscriptases, and the like. Preferably, whenever the kit is for PCRamplification, the nucleic acid polymerase is Taq polymerase, e.g. asdisclosed by G U.S. Pat. No. 4,889,818. Guidance for selecting a PCRreaction buffers and nucleoside triphosphate mixes for particularembodiments can be found in innis et al, Editors, PCR Protocois: A Guideto Methods and Applications (Academic Press, New York, 1990). A typical10×PCR reaction buffer comprises 15 mM MgCl₂, 500 mM KCl, and TrHCl, pH8.3.

Preferably, whenever the kit permits a ligase based amplificationreaction, e.g. as disclosed by Landegren et al, U.S. Pat. No. 4,988,617or the like, the nucleic acid ligase is a thermostable ligase, such asdisclosed by Barany, Proc. Natl. Acad. Sci., Vol. 88, pgs. 189-193(1991). Guidance for selecting a ligase-based reaction buffer can befound in Landegren et al (cited above), Wu et al, Genomics, Vol. 4, pos.5W-569 (1989): Barany (cited above), and Nickerson et al, Proc. Natl.Acad. Sci., Vol. 87, pgs. 8923-8927 (1990). A typical ligation reactionbuffer comprises 20 mM TrisHCl, pH 7.6; 50 mM KCl; 10 mM MgCl₂; 1 mMEDTA; 10 mM NAD⁺, and 10 mM dithiothreitol.

The dye-labelled oligonudeotides of the kit can have a wide range oflengths, but preferably their length are in the range of 6 to 60nucleotides. More preferably, the oligonucleotides for ligation kits arein the range of 6 to 30 nudeotides in length, and most preferably, theoligonudeotides for ligation kits are in the range of 16 to 25nucleotides in length. The particular nucieotide sequence of theoligonucleotides are, of course, dictated bythe target sequences soughtto be amplified. In embodiments for PCR amplification, selection ofoligonudeotides for use as PCR primers is well known in the art, e.g.Innis et al (cited above), Hiller and Green, PCR Methods andApplications, Vol. 1, pgs. 124-128 (1991), and the like.

Preferably, in kits for DNA sequencing wherein dye-terminators areprovided, each dideoxynucdeoside triphosphate is separately labelledwith a dye selected from the set comprising 5- and 6-carboxyfluorescein,5- and Scarboxy-4.7-dlchlorofluorescein, 2′,7′-dimethoxy-5- and6-carboxy-4,7-dichlorofluorescein, 2′,7′-dimethoxy-4′,5′-dichloro-4′,5′-and 6-carboxyfluorescein, 2′,7′-dimethoxy-4′,5′-dichloro-5- and6-carboxy-4,7-dichloofluorescein, 1′,2′,7′,8′-dibenzo-5- and6-carboxy-4,7dichlorofluorescein, 1′,2′,7′,8′-benzo-4′,5′-dichloro-5-and 6-carboxy-4,7-dichlorofluorescein, 2′,7′-dichloro-5- and6-carboxy-4,7-dichlorofluorescein. and 2′,4′,5′,7′-tetrachloro-5- and6-carboxy-4,7-dichlorofluorescein. More preferably. dideoxythymidinetriphosphate is labelled with 6-carboxyfluorescein (“6-FAM”),dideoxycytidine triphosphate is labelled with2′,4′,5′,7′-tetrachloro-5crboxyfluocein (“5-ZOE”), dideoxyadenosinetriphosphate is labelled with2′,4′,5′,7′-tetrachloro-4,7dichloro-5-carboxyfluorescein (“5-HEX”), anddideoxyguanosine triphosphate is labelled with1′,2′,7′,8′-dibenzo-4,7-dichloro-7-carboxyfluoressein (“5-NAN”). it isunderstood that dideoxyadenosine includes 2′,3′-dideoxy-7deazaadenosineand dideoxyguanosine includes 1′,3′-dideoxy-7-deazaguanosine and2′3′-dideoxy-7-deazainosins, and dideoxythymidine includes2′,3′-dideoxyuridine. Usually, the dideoxynudeoside triphosphates arelabelled by way of a linking group. Preferably, the linking group linksa 5 carbon of the 2′,3′-dideoxycytidine or 2′,3′-dideoxyuridine to a 5or 8 carbon of a dye, and the linking group links a 7 carbon of the2′,3′-dideoxy-7-deazaadenosine or 2′,3′-dideoxy-7-guanosineor2′,3′-dideoxy-7-deazainosine to a 5 or 6 carbon of a dye. Preferably,the linking group is carboxyaminoalkynyl, and most preferably, thelinking group is 3carboxyamino-1propynyl.

Preferably, in kits for DNA sequencing wherein dye-temoinators areprovided, the nucleic acid polymerase is Sequenase™.

EXAMPLE 1 4,7-dichloro-5-(and 6-)carboxyfluorescein (“ALF”)

0.58 g of 3,6-dichlonttrimellitic acid, 0.72 g of resorcinol, 0.5 mlconcentrated suifuti acid, and 3 ml of propionic acid were refluxed 12hours under argon. The reaction mixture w8 poured into 150 ml water; theprecipitate was dried, taken into 3 ml pyridine and acetylated with 2 mlacetic anhydride for 1 hour. The acetylation mbiture was taken into 100ml ethyl acetate, washed with 1 N hydrochloric acid, water, andevaporated to dryness. The residue was placed on 15 grams of silica geland eluted with 50 ml ethyl acetate, then 4:1 ethyl acetate:methanol.Fractions containing UV active material with R_(f) of about 0.2 (4:1ethyl acetate:methano/silica gel) were evaporated to dryness. Thisresldue was dissolved in 10 ml methanol and then 1 ml of 4 N sodiumhydroxide was added. After 10 minutes, the reaction mixture was dilutedto 200 ml with water and then 0.5 ml of concentrated hydrochloric acidwas added. The total mixture was extracted with 200 ml of ethyl acetate,after which the ethyl acetate was dried with sodium sulfate andevaporated to dryness yielding 102 mg of yellow-green solid.

EXAMPLE 2 4,7-dichloro-5-(and 6-) carboxvfluomscein N-hvdroxvsuccinimide(NHS) ester

13.7 ng of fluorescein from Example 1, 3.3 mg of 30 NHS, 6,4 mg DCC and1 ml ethyl acate were stired 0.5 hours. The solid was filtered, and thesupematnt was washed three times with 1:1 bine:water, dried with sodiumsulfate, and evaporated to dryness yielding 15 mg of NHS ester.

EXAMPLE 3 Conjugation of 4,7-dichloro-5-(and 6-)carboxyfluorescein withaminoalkyloligonucleotides

5 mg of NHS ester from Example II were dissolved 5 in 20 ul of DMSO: 3ul of this solution were added to a solution consisting of 20 ul of 1.0mM 5′-aminophosphate oligonucleotide (an 18-mer) in water and 10 ul of 1M sodium bkarbond/sodium carbonate buffer. pH 9.0. After one hour in thedark, the solution was passed through a 10 ml Sophedox G 25 (medium)column with 0.1 M triethylammonium acetate buffer, pH 7.0. The band ofcolored material eluting in the exclusion volume was collected. Reversephase HPLC showed two major fluent peaks, corresponding to the 5- and6-isomers of the dye conjugated onto the DNA. The peaks were collected,and the fluorescence speera in 50% urea at pH 8.0 showed full width athalf m of 34 nm with the emission maxima at 528 nm.

EXAMPLE 4 2′,7′-dimethoxy-5-(and 6-)carboxy 4,7-dichlorofluorescein(“BUB”)

The procedure of Example I was followed except that the following metalsand quantities were substituted: 1.47 g 4-methylresorcinol, 0.60 g of3,8-dichlorimellitic ad., 0.2 ml concentrated sulfuric acid, and 4 mlpropionic acid. The procedure yielded 0.180 g of4,7-dichloro-2′,7′-dimethoxy-5-(and 6-)carboxyfluorescein.

EXAMPLE 5 2′,7′-dimethoxy-5-(and 6-)carboxy 4,7-dichlorofluorescein NHSester

18 mg of this dye NHS ester were prepared as in Example II using 18 mgof dye from Example IV. 3.5 mg NHS, 8.4 mg DCC, and 2 ml ethyl acetate.

EXAMPLE 6 Conjugation of 4,7-dichloro-2′,7′-dimethoxy 5-(and6-)carboxyfluorescein with amino-alkyloligonucleotide

The procedure of Example III was followed using the dye NHS ester ofample V. The fluorescence aspect of the two peaks collected duringreverse phase HPLC showed full widths at half max of 37 nm with emissionmaxima at 544 nm in 50% urea at pH 82.

EXAMPLE 7 2′7′-dimethoxy-4′,5′-dichloro-5-(and6-)carboxy-4,7-dichlorofluorescein (“LOU”)

This dye was prepared from the dye of Example IV and sodium hypochie inaqueous sodium hydride.

EXAMPLE 8 4,7-dichloro-2′,7′-dimethoxy-4′,5′-dichloro-5-(and6-)carboxyfluorescein NHS ester

1.1 mg of this dye NHS ester was prepared from 0.7 mg of the dye fromExample VII, 0.45 mg of NHS, 0.7 ma DCC, and 0.2 ml ethyl acetate as inExample II.

EXAMPLE 9 Conjugation of 4,7-dichloro-2′,7′-dimethoxy4′,5′-dichloro-5-(and 6-)carboxyfluorescein withaminoalkyloligonucleotides

The dye nucleotide conjugate of this example was prepared as in ExampleIII using tho dye NHS ester from Example VIII. The fluorescence spectraof the two peaks collected during revere phase HPLC showed full widthsat half max of 38 nm with emulsion maxima at 558 nm in 50% urea at pH8.2.

EXAMPLE 10 1′,2′,7′,8′-dibenzo-5-(and 6-)carboxy-4,7-dichlorofluorescein(“NAN”)

First 3,6-dichlorotrimellitic acid trichloride was prepared: A mixtureof 0.5 g of 3,6-dichloromellitic acid and 1.3 g of phosphorouspentachloride was heated at 130° C. for 40 minutes. The mixture wascooled to room temperature and poured into ice. The mixture was thenextracted with 40 ml ether, the organic fraction was washed twice with15 ml water, dried with MgSO₄, and concentrated to a clear oil (0.7 g).The acid trichloro was used without further purification. NAN wasprepared as follows: A mixture of 2,7 g of 1,3-dihydroxynaphthalene,2.84 g of 3,6dichlorotrimellitic acid trichloride, and 8 ml of propionicadd was refluxed for 2 hours. Water (50 ml) and ethyl acetate (50 ml)were added. The layers were separated and the organic layer wasextracted three times with 50 ml of 1 M NaHCO₃. The aqueous solution washeated to boiling and acidified with concentrated HCl. The resulting redsolid (0.2 0) was filtered and dried.

EXAMPLE 11 1′,2′,7′,8′-dibenzo-4′,5′-dichloro-5-(and6-)carboxy-4,7-dichlorofluorescein (“DEB”)

20 mg of NAN. sodium hydroxide (34 ul of a 15% solution), water (1 ml),and sodium hypochlorite (170 ul of a 5% solution) were combined. Reversephase HPLC showed 92% reacion. The solution was acidified with HCI.extracted with 20 ml of ethyl acetate. dred (Na₂SO₄), and concentratedto 20 mg. The solid was purified by chromatography on a silica gelcolumn (1″ diameter×2″ height), eluting with 800:60:18 methylenechloride:methanol:acetic acid. The dye solution was concentrated, anddilute HCl and ethyl acetate added. The organic phase was dried (MgSO₄)and concentrated to 20 ng of DEB.

EXAMPLE 12 Formation of 1′,2′,7′,8′-dibenzo-5-(and6-)carboxy-4,7-dichlorofluorescein NHS ester

NAN (10 mg) was dissolved in 2 ml of ethyl acetate, and NHS (10 mg) andDCC (5 mg) was added. After 20 minutes, the solution was dark red incolor and a crystalline sold appeared.

Thin layer Schromatography on a silica gel using 800:80:16 methylenechloride:methanol:acetic acid showed complete conversion to the NHSester. The ethyl acetate solution was wished with dilute HCl, dried(NaSO₄) and concentrated to a red solid (15 mg).

EXAMPLE 13 Using ALF-, BUB-, LOU-, and NAN-oligonucleotide conjugates asdye-labeled primers in DNA sequence analysis

An all-fluorescein set of dyes was used to label DNA fragments in thechain termination approach employing the Applied Biosystems (FosterCity. Calif.) Model 370A automated DNA sequencer. The manufacturersprotocol (User Bulletin DNA Sequencer Model 370, issue No. 2, Aug. 12,1987), which is incorporated by reference) was followed foramplification of the unknown DNA in M13 and preparation of separatelylabeled DNA fragments for gel electrophoretic separation. Dye-labeledprimers were prepared as described in the examples above. That is, NHSester of the respective dyes were prepared and reacted with the5′-aminohexyl-derivatized M13 universal primer(5′-TCCCAGTCACGACGTTGT-3′) to form the dye-labeled primers for the fourseparate dideoxy reaction mixtures. The following modifications weremade to the standard protocol: 5-carboxy-4,7-dichlorofluorescein labeledthe primer in the dideoxycytidine reaction,2′,7′-dimethoxy-5-carboxy-4,7-dichlorofluorescein labeled the primer inthe dideoxyadenosine reaction2′,7′-dimethoxy-4′,5′-dichloro-6-carboxy-4,7-dichlorofluorescein labeledthe primer in the dideoxyguanosine reaction,1′,2′,7′,8′-dibenzo-4,7-dichlorofluosceins labeled the primer in thedideoxythymidine reaction, labeled DNA fragments from the respectivereactions were combined in the following molar ratios for loading ontothe gel: 1:1:4:2 ddC reaction:ddA reaction:ddG reaction:ddT reaction,and detection was accomplished with a modified filter wheel using 10-nmbandpass filters centered at 535, 550, 565, and 580 nm.

EXAMPLE 14 Using ALF-, BUB-, DEB and NAN-oligonucleotide conjugates asdye-labeled primers in DNA sequence analysis

The same procedure was followed as described for Example XIII, exceptfor the following: (i)1′,2′,7′,8′-dibenzo-4′,5′-dichlorocarboxy-4,7-dichlorofluorosceinlabeled the primer in the dideoxyguanosine reaction, (ii) labeled DNAfragments from the respective reactions were combined in the followingmolar ratios for loading on the gel: 1:1:2:15 ddC reaction:ddAreaction:ddG reaction:ddT reaction, and (ii) 5 nm bandpass filters worecentered at 540, 580, 580, and 610 nm.

EXAMPLE 15 2′,7′-dichloro-5-(and 6)-carboxy-4,7-dichlorofluoresein(“5-(and 6-)TET”)

A mixture of 4-chlororesorcinol (10 g), 4,7-dichlorotrimellitic acid (10g), and methanesulfonic acid (30 mL) were combined and heated to 140°C.-150° C. for two hours. The red mixture was poured into water (100 mL)and extracted with ethyl acetate (100 mL). The organic phase was washedtwice with dilute aqueous HCl and concentrated to go brown solid (19 g).Pyridin (40 mL) and acetic anhydride (10 mL) were added to the solid andthe mixture refluxed for 0.5 hours. The solution was allowed to cool for1 hour at 4° C.

Crystals were separated by function to yield a white solid (5.4 ).Hydrolysis of a small portion (by addition of 0.02 mL of 0.1 N NsCl and0.02 mL of ethanol to 2 ng solid) followed by analysis on reverse phaseHPLC showed that the solid contained a 92:8 ratio of isomers (6-carboxyTET:5carboxy TET ). A second recrystallization provided nearlyisometrically pure dye as the diacetate (99:1 ratio). 5-TET can berecovered from the filtrate by hydrolysis of the discelate form of 5-TETfollowed by recrystallization from acetonitrile.

Sodium hydroxide (3 g) and water (10 mL) were added to 8TET diacetate(8.8 g)(obtained as the first of two peaks off the HPLC column).Additional water (50 mL) was added until the solution becamehomogeneous. To the dark red solution was added concentrated HCl (15mL). A yellow precipitated formed. The mixture was extracted with ethylacetate (100 mL). The organic layer was concentrated to pale yellow,nearly colorless solid (7.4 g of 6-TET).

EXAMPLE 16 2′,4′,5′,7′-tetrachloro-5-(and6-)carboxy-4,7-dichlorofluorescein (“5- and 6-HEX”)

To a 1-liter Erienmeyer flask equipped with a magnetic stirring bar wasadded 5- or 8-TET (6.3 ) and 1 M bonate buffer at pH 9.4 (60 mL).Household bleach (sodium hypochlorite, 50 ml) was added dropwise over 20minutes. The progress of the reaction was monitored by reverse phaseHPLC. A total of 67 mL of bleach was added. The solution was acidifiedwith concentrated HCl (15 mL) and extracted with ethyl acetate (100 mL).The organic phase was concentrated to a bright yellow solid (7.3 g). ¹HNMR (DMSO-d₆) δ8.1(1H); 7.4 (2H).

1. In a method of detecting a plurality of electrophoretically separatedclasses of DNA fragments, an improvement comprising labelling DNAfragments of at least one class with a 4,7-dichlorofluorescein dye. 2.The method of claim 1 wherein said 4,7-dichlorofluorocein is difined bythe formula:

wherein: A′ is hydrogen, fluoro, chloro, a linking functionality, or agroup that may be converted to a linking functionality; B′ is fluoro,chloro, or an acidic anionic group; X′ is hydrogen, fluoro, or chloro;Z₁ is hydrogen or, when taken with Z₂, benzo; Z₂, when taken alone, ishydrogen, halo, lower alkyl, lower alkyloxy, a linking functionality, ora group that may be converted to a linking functionality, or when takenwith Z₁, benzo; Z₃ and Z₄ are separately hydrogen, halo, lower alkyl,lower alkyloxy, a linking functionality, or a group that may beconverted to a linking functionality; Z₅, when taken alone, is hydrogen,halo, lower alkyl, lower alkyloxy, a linking functionality, or a groupthat may be converted to a linking functionality, or when taken with Z₆,benzo; Z₆ is hydrogen or, when taken with Z₅, benzo; and wherein atleast one of A, Z₂, Z₃, Z₄, and Z₅ is a linking functionality or a groupthat may be converted to a linking functionality.
 3. The method of claim2 wherein: A′ is carboxyl, sulfonyl, isothlocyanato, succinimidylcarboxytate, phosphoramidite, or amino; B′ is carboxyl or sulfonyl; X′is hydrogen or chloro; Z₂, when taken alone, is hydrogen, methyl, ethyl,methoxy, ethoxy, or chloro; Z₃, and Z₄ are separately hydrogen, methyl,ethyl, methoxy, ethoxy, chloro, carboxyl, sulfonyl, isothiocyanate,succinimidyl carboxylate, phospboamidite, or methylamino: Z₅, when takenalone, is hydrogen, methyl, ethyl, methoxy, ethoxy or chloro; andwherein only one of A′, Z₃, and Z₄ is carboxyl, sulfonyl, metylamino,isothiocyanate, succinimidyl carboxylate, phosphoramidite, or amino. 4.The method of claim 3 wherein Z₃, and Z₄ are separately hydrogen,methyl, ethyl, methoxy, ethoxy, or chloro.
 5. The method of claim 4wherein Z₂, when taken alone, is hydrogen, methoxy, ethoxy, or chloro;Z₃, and Z₄ are separately hydrogen, methoxy, ethoxy, chloro; and Z₅ whentaken alone, is hydrogen, methoxy, ethoxy, or chloro.
 6. The method ofclaim 5 wherein B′ is carboxy and A′ is carboxy, succinimidyl,carboxylate, or phosphoramidite.
 7. A compound having the formula:

wherein A′ is hydrogen, fluoro, chloro, a linking functionality or agroup that may be converted to a linking functionality; B′ is fluoro,chloro, or an acidic anionic group; X′ is hydrogen, fluoro, or chloro;Z₃ and Z₄ are separately hydrogen, halo, a linking functionality, or agroup that may be converted to a linking functionality; and wherein atleast one of A′, Z₃, and Z₄ is a linking functionality or a group thatmay be converted to a linking functionality.
 8. The compound of claim 7wherein A′ is carboxyl, sulfonyl, isothiocyanate, succinimidylcarboxylate, phosphoramidite, or amino: B′ is carboxyl or sulfonyl: X′is hydrogen; Z₃ and Z₄ are separately hydrogen, halo, carboxyl,sulfonyl, or methylamino.
 9. The compound of claim 8 wherein only one ofA′, Z₃, and Z₄ is carboxyl, sulfonyl, methylamino, or amino.
 10. Thecompound of claim 9 wherein A′ and B′ are carboxyl, Z₃ is hydrogen orchloro, and Z₄ is hydrogen or chloro.
 11. A kit for detecting aplurality of electrophoretically separated classes of DNA fragmentscomprising an oligonucleotide labelled with a 4,7-dichlorofluoresceindye.
 12. The kit of claim 11 further compounding: an enzyme selectedfrom the group consisting of acid polymerase and nucleic acid ligase;and a reaction buffer.
 13. The kit of claim 12 wherein said enzyme is anucleic acid polymerase and wherein said kit further includes anucleoside triphosphate mix.
 14. The kit of claim 12 wherein said enzymeis a nucleic acid ligase.
 15. A kit for sequencing DNA comprising: anoligonucleotide with a 4,7-dichlorofluorescein dye; a nucleic acidpolymerase; a reaction buffer; and a nucleoside triphosphate mix. 16.The kit of claim 15 wherein said 4,7-dichlorofluorescein dye is definedby the formula:

wherein: A′ is hydrogen, fluoro, chloro, or a group that may beconverted to a linking functionality; B′ is fluoro, chloro, or an acidicanionic group; X′ hydrogen, fluoro, or chloro; Z₁ is hydrogen or, whentaken with Z₂, benzo; Z₂ when taken alone, is hydrogen, halo, loweralkyl, lower alkyloxy, or a group that may be converted to a linkingfunctionality, or when taken with Z₁, benzo; Z₃ and Z₄ are separatelyhydrogen, halo, lower alkyl, lower alkyloxy, or a group that may beconverted to a linking functionality: Z₅, when taken alone, is hydrogen,halo, lower alkyl, lower alkyloxy, or a group that may be converted to alinking functionality, or when taken with Z₆, benzo; Z₆ hydrogen or,when taken with Z₅, benzo; and wherein at least one of A′, Z₂, Z₃, Z₄,and Z₅ is a group that may be converted to a linking functionality. 17.The kit of claim 16 wherein: A′ and B′ are carboxy; X′ hydrogen orchloro; Z₂, when taken alone, is hydrogen, methyl, ethyl, methoxy,ethoxy, or chloro Z₃, and Z₄ are separately hydrogen, methyl, ethyl,methoxy, ethoxy, chloro; and Z₅, when taken alone, is hydrogen, methyl,ethyl, methoxy, ethoxy, or chloro.
 18. A kit for sequencing DNAcomprising: a dye-terminator mix wherein at least one dye-terminator islabelled with a 4,7-dichlorofluorescein dye; a nucleic acid polymerase;a nucleoside triphosphate mix; and a reaction buffer.
 19. The kit ofclaim 18 wherein said dye-terminator mix comprise dideoxynudeotidetriphosphates selected from the group consisting of dideoxyadenosinetriphosphate, dideoxycytidine triphosphate, dideoxyguanosinetriphosphate, and dideoxythymidine triphosphate wherein each of saiddideoxynudeotide triphosphates is separately labelled with a dye3elected from the group consisting of 5- and carboxyfluorescein. 5- andcarboxyl-4,7-dichlorofluorescein, 2′,7′-dimethoxy-5- andcarboxy4,7-dichlorofluorocein, 2′,7′-dimethoxy-4′,5′-dichloro-5- and6-carboxyfluorescein. 2′,7′-dimethoxy-4′,5′-dichloro-5- and6-carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-5- and6-carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-4′,5′-dichloro-5-and 6-carboxy-4,7dichlorofluorescein, 2′,7′-dichloro-5- and6-carboxy-4,7-dichlorofluorescein, and 2′,4′,5′,7′-tetrachloro-5- and6-carboxy-4,7-dichlorofluorescein.
 20. The kit of claim 19 wherein saiddideoxythymidine triphosphate is labelled with 6-carboxyfluorescein,said dideoxycytidine triphosphate is labelled with2′,4′,5′,7′-tetrachloro-5-carboxyfluorescein, said dideoxyadenosinetriphosphate is labelled with2′,4′,5′,7′-tetrachloro-4,7-dichloro-5-carboxyfluorescein, and saiddideoxyguanosine triphosphate is labelled with1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein.
 21. The kit ofclaim 20 wherein said nucleic acid polymerase is Sequenase™.